ABSTRACT
Objective:To evaluate the role of spinal mammlian target of rapamycin (mTOR)/ribosomal S6 kinase 1 (S6K1)/glioma associated oncogene homolog 1 (Gli1) signaling pathway in chronic morphine tolerance in mice.Methods:Healthy male Kunming mice, aged 8-10 weeks, weighing 23-25 g, were used in the study.The experiment was performed in two parts.Experiment I Fifty mice were randomly assigned into 2 groups: normal saline group (group S, n=10) and morphine group (group M, n=40). In M and S groups, morphine and normal saline 10 mg/kg were injected subcutaneously, respectively, twice a day for 7 consecutive days.The thermal pain threshold (TPT) was measured and the maximum analgesic effect percentage (MPE) was calculated at 1 day before administration and 30 min after the last administration every day.Ten mice in each group were randomly selected and sacrificed after measurement of TPT at 1, 3, 5 and 7 days after administration in group M and after the last measurement of TPT in group S, and the lumbar segment (L 4-6) of the spinal cord was removed.Experiment Ⅱ Forty mice were randomly divided into 4 groups ( n=10 each): KU-0063794+ morphine group (group KU+ M), dimethyl sulfoxide (DMSO)+ morphine group (group DM+ M), morphine+ KU-0063794 group (group M+ KU) and morphine + DMSO group (group M+ DM). Morphine 10 mg/kg was injected subcutaneously twice a day for 7 consecutive days in 4 groups.At 1-3 days of morphine injection, mTOR specific inhibitor KU-0063794 200μl (1 μg/μl) and 10% DMSO 200 μl was injected intraperitoneally in KU+ M group and DM+ M group at 30 min before administration twice a day.At 5-7 days of morphine injection, KU-0063794 200μl (1 μg/μl) or 10% DMSO 200 μl was injected intraperitoneally in group M+ KU or group M+ DM at 30min before administration, respectively, twice a day.TPT was measured and MPE was calculated at 1 day before morphine injection and at 30 min after the last administration every day.The animals were sacrificed after the last measurement of TPT, and the lumbar segment (L 4-6) of the spinal cord was removed for determination of the expression of spinal mTOR, phosphorylated mTOR (p-mTOR), S6K1, phosphorylated S6K1 (p-S6K1) and Gli1 (using Western blot). Results:Experiment Ⅰ Compared with group S, MPE was significantly increased at each time point after administration at 3, 5 and 7 days after administration, expression of spinal p-mTOR, p-S6K1 and Gli1 was significantly down-regulated ( P<0.05), and no significant change was found in mTOR and S6K1 in group M ( P>0.05). Experiment Ⅱ Compared with group DM+ M, MPE was significantly decreased at 3-7 days after morphine injection, expression of p-mTOR, p-S6K1 and Gli1 in spinal cord was down-regulated ( P<0.05), and no significant change was found in expression of mTOR and S6K1 in group KU+ M ( P>0.05). Compared with group M+ DM, MPE was significantly increased at 6-7 days after morphine injection, expression of p-mTOR, p-S6K1 and Gli1 in spinal cord was down-regulated ( P<0.05), and no significant change was found in mTOR and S6K1 in group M+ KU ( P>0.05). Conclution:Spinal mTOR/S6K1/Gli1 signaling pathway is involved in the development and maintenance of chronic morphine tolerance in mice.
ABSTRACT
To investigate the potential use of suicide gene systems in vascular targeting gene therapy, HSV-tk/GCV and EC-cd/5-FC systems were established on vascular endothelial cells in vitro by adenovirus transduction. Both modified cell lines were highly sensitive to prodrugs, the IC_(50) for GCV was 0.2?mol/L in IGll/Ad-tk cells, and IC_(50) for 5-FC was 20?mol/L in IGll/Ad-cd cells, while the parental endothelial cells were insensitive even at the highest concentrations of prodrugs in the experiment. Mixed cellular assay showed that significant bystander killing effect was exhibited in modified endothelial cells. Also, the apoptosis involved in these prodrug-mediated growth inhibitions has been shown under electron microscopy. These results indicated that both HSV-tk/GCV and EC-cd/5-FC systems could efficiently suppress vascular endothelial cell growth in vitro , suggesting the feasibility of using suicide gene in vascular targeting gene therapy.